Common wheat represents a typical allohexaploid species and provides a good experimental system for studying genomic alterations associated with allopolyploidization. We studied three homoeologous loci of wheat Wknox1 gene, which is highly homologous to the knotted1 (kn1)-like homeobox (KNOX) genes functioning at shoot apical meristems (SAM). Wknox1 transcripts were detected in SAM, and its overexpression caused abnormal leaf morphology with occasional ectopic leaves in transgenic tobacco plants. A comparative study of the three Wknox1 genomic sequences revealed accumulation of a large number of mutations including insertions and deletions, particularly in the fourth intron and the 5'-upstream region. Some structural mutations including MITE-insertions were allocated in the evolutionary lineage of the wheat genome, suggesting that they occurred at all stages of wheat evolution. A mutation rate was the highest in the Wknox1b locus, which is consistent with the known highest degree of differentiation in the B genome. Despite the structural differentiation, all three Wknox1 homoeologs showed an identical expression pattern in wheat and their promoter regions induced the conserved expression pattern in transgenic tobacco plants. A potential of the intragenic diversity in homoeologs of essential genes as a tool for studying the genome evolution associated with allopolyploidization was discussed.

Introgression between related species with different ploidy levels has played important roles in wheat subspecies differentiation. Persian wheat, a cultivated tetraploid wheat subspecies (Triticum turgidum subsp. carthlicum), is postulated to have evolved through interploidy hybridization between tetraploid and hexaploid wheats. Here, we report evidence for the origin of subsp. carthlicum based on the discovery of a new allele for the 5th-to-6th exon region of the Wknox1bKNOTTED1-type homeobox gene in a common wheat subspecies (T. aestivum subsp. carthlicoides). In this Wknox1b region, subsp. carthlicoides contains an inverted duplication mutation in the 3' flanking region of a 157-bp MITE insertion site. This structural mutation resulted in the suppression of Wknox1b expression in subsp. carthlicoides, but no structural mutation was observed in the same region of subsp. carthlicum. In addition, the carthlicum allele for the Wknox1b 5th-to-6th exon region exhibited the same sequence as that in the wild emmer wheat subsp. dicoccoides. These observations support an alternative hypothesis that subsp. carthlicum evolved by interploidy hybridization between subsp. carthlicoides and tetraploid wheat.

Maize rough sheath2 (RS2) and Arabidopsis ASYMMETRIC LEAVES1 (AS1) both encode a Myb transcription factor and repress Knotted1-type homeobox (KNOX) genes. The RS2/AS1-KNOX relationship is functionally conserved between maize and Arabidopsis. Here, we cloned wheat orthologs of RS2/AS1 and of a maize rough sheath1 (rs1) KNOX gene and named them WRS2 and WRS1, respectively. WRS1 mRNA was detected at leaf insertion points of the vegetative shoot meristem but was missing in differentiating floral organs. Conversely, WRS2 transcripts accumulated in initiating and developing floral organs. Transgenic tobacco plants expressing WRS1 showed morphological alterations typically observed due to expression of other KNOX genes. WRS2 with a deletion of the Myb domain could interact with NtPHAN to form a heterodimer, and expression of the truncated WRS2 gene conferred a dominant-negative phenotype similar to that expected and induced ectopic expression of an endogenous KNOX gene. Moreover, WRS2 expression alleviated morphological alterations in tobacco plants expressing the wheat KNOX gene. Therefore, the WRS2 gene product represses KNOX expression. These results indicate that the WRS2-KNOX relationship plays a fundamentally important role in lateral organ initiation and differentiation of meristems in wheat development. The antagonistic relationship between WRS2 and KNOX around meristematic tissues has been functionally conserved during wheat evolution.